Ketol fatty acid derivative and plant growth regulating agents

ABSTRACT

α-Ketol unsaturated fatty acid derivatives represented by the following formula (1), α-ketol unsaturated fatty acid amide derivatives represented by the following formula (6), and plant growth regulators containing, as the active ingredient, at least one of these derivatives: 
                 
 
wherein R 1  represents linear C 1-5  alkyl; R 2  represents hydrogen, etc.; R 3  represents hydrogen, etc.; R 4  represents hydrogen, etc.; R 5  represents hydrogen, etc.; n is an integer from 3 to 15; and the carbon atoms at the A- and B-positions may be in either (R)- or (S)-configuration; and 
                 
 
wherein R 7  represents C 1-17  alkyl, etc; R 8  represents hydrogen, etc.; R 9  represents α-ketolalkylene; and R 10  represents specific divalent linear hydrocarbyl.

This application is a 371 of PCT/JP01/02328 Mar. 23, 2001, which claimsthe foreign priority under 35 U.S.C. § 119(a)-(d) to Japan 2000-128625Mar. 25, 2000, Japan 2000-361216 Nov. 28, 2000, and Japan 2000-61528Mar. 6, 2001.

TECHNICAL FIELD

The present invention relates to a specific class of α-ketol fatty acidderivatives and to a process of preparing the derivatives. Moreover, thepresent invention relates to plant growth regulators making use of theplant growth promoting effect of the derivatives.

BACKGROUND ART

Development of technology to regulate plant growth is a key issue inpromoting the supply efficiency of crop plants and horticultural plants.

Among a variety of effects manifested by the regulation of plant growth,promotion of plant growth is a typical effect. Conceivable factors thatdetermine the growth rate of plants include temperature, light, andnutrients. From the past, in order to promote growth of a plant,attempts have been made to select appropriate temperature and lightconditions in accordance with the properties of the plant of interest.In addition to such selection of temperature and light conditions,application of a fertilizer is also a well-established technique and hasprovided a certain effect.

However, effects yielded by application of a fertilizer are limited;i.e., an increased amount of fertilizer does not necessarily attain agrowth promotion effect commensurate with such an increase. Contrary toexpectations, excessive application of a fertilizer not only hampers thegrowth of the plant, but possibly raises the risk of pollution of soil.

In particular, in an early stage of plant growth, application of afertilizer tends to induce nutritional disorder; therefore, applicationof a fertilizer is typically withheld in this stage.

Therefore, there exists demand for a compound which exhibits plantgrowth promoting effect and is quite different from conventionalfertilizer composed of nitrogen, phosphate, potassium, etc.

There also exists demand for improvement in supply efficiency of cropplants or horticultural plants, through discovery of means forregulating, in accordance with needs, growth of plants, on the basis ofvarious effects, including not only such promotion of plant growth, butalso promoted formation of flower buds, retarded aging of plants,prevention of dormancy of plants, and mitigation of plant stresses suchas drying and high temperature.

In relation to the above, the present inventors previously found that acertain class of ketol fatty acids exhibits the flower bud formationpromoting activity for a broad range of plants (see, for example,Japanese Patent Application Laid-Open (kokai) No. 11-29410). Now, theinventors are in the course of clarifying that such specific ketol fattyacids are intimately involved in various life-related phenomena ofplants.

Thus, a primary object of the present invention is to create, as anextension of the mentioned findings regarding the ketol fatty acids, anovel substance which meets the aforementioned subject matter, and toprovide a novel plant growth regulator containing the novel substance.

DISCLOSURE OF THE INVENTION

The present inventors have carried out careful studies so as to attainthe above object, and quite surprisingly, have found that an α-ketolunsaturated fatty acid derivative, which is formed through bonding of anα-ketol unsaturated fatty acid having a specific structure tonorepinephrine (which is one form of catecholamine), and an α-ketolunsaturated fatty acid amide derivative having a specific structure areendowed with excellent plant growth regulating effects, leading tocompletion of the invention.

Accordingly, in the present application, the inventors provide thefollowing inventions.

A first invention is drawn to an α-ketol unsaturated fatty acidderivative represented by formula (1) (hereinafter the derivative may bereferred to as ketol fatty acid derivative I of the invention):

wherein R¹ represents linear C₁₋₅ alkyl; R² represents hydrogen,hydroxyl, methyl, or ethyl; R³ represents hydrogen or carboxyl; R⁴represents hydrogen, methyl, ethyl, isopropyl, acetyl, or tert-butyl; R⁵represents hydrogen, hydroxyl, methoxy, ethoxy, formyl, orhydroxymethyl; n is an integer from 3 to 15; and the carbon atoms at theA- and B-positions may be in either (R)- or (S)-configuration.

A second invention is drawn to a process of preparing ketol fatty acidderivative I of the invention comprising the steps of mixing an α-ketolunsaturated fatty acid represented by formula (2) (hereinafter the fattyacid may be referred to as α-ketol unsaturated fatty acid (2)) with acatecholamine represented by formula (3) or a derivative thereof(hereinafter may be referred to as catecholamine (3)) and incubating theresultant mixture under neutral to basic conditions, to thereby yield anα-ketol unsaturated fatty acid catecholamine derivative (hereinafter theprocess may be called the process of the invention):

wherein R¹ and n have the same meanings as defined in relation toformula (1) above, and

wherein R² to R⁵ have the same meanings as defined in relation toformula (1) above, and R⁶ represents hydrogen, linear or branched alkyl,or acyl.

A third invention is drawn to an α-ketol unsaturated fatty acid amidederivative represented by formula (6) (hereinafter the derivative may bereferred to as ketol fatty acid derivative II of the invention):

wherein R⁷ represents C₁₋₁₇ alkyl or C₂₋₁₇ unsaturated hydrocarbylhaving 1 to 5 double bonds (wherein the number of double bonds does notexceed the total number of carbon-carbon bonds); R⁸ represents hydrogenor a side chain of α-amino acid; R⁹ represents α-ketolalkylene; and R¹⁰represents a divalent linear hydrocarbyl of the following formula:—(CH₂)_(L)—(HC═CH)_(m)—(CH₂)_(k)—wherein L, m, and k are integers, with m being 0 or 1 and L+k being 1 to15.

A fourth invention is drawn to a plant growth regulator containing ketolfatty acid derivative I and/or II of the invention as an activecomponent (hereinafter the plant growth regulator may be referred to asthe plant growth regulator of the invention or the present plant growthregulator). The present plant growth regulator may assume a variety offorms (such as a flower bud formation promoter, a plant activator, aplant growth promoter, an aging retardant for plants, a flower seasonextender, a plant dormancy inhibitor, and a plant stress suppressor) onthe basis of individual effects of the ketol fatty acid derivative Iand/or II of the invention on plants.

As used herein, the expression “plant growth regulation” meansregulation, in any form, of life-related activities of a plant, and itsconcept broadly encompasses not only a variety of plant activatingactions, such as plant growth promotion, anti-aging, prolonging offlower season, dormancy inhibition, and imparting of stress resistanceto plants, but also promotion of flower bud formation.

Also, the concept of “plant growth promotion” broadly encompassespromoting enlargement of stems and leaves, growth promotion of tubersand root tubers, promotion of fruit bearing, fruit growth promotion,etc.

The present inventors had previously found that the aforementionedα-ketol unsaturated fatty acid (2), alone or in combination withnorepinephrine, one form of catecholeamine (3), exhibits flower budformation promoting activity for a broad range of plants, and haddisclosed this in the aforementioned Japanese Patent ApplicationLaid-Open (kokai) No. 11-29410. However, the ketol fatty acidderivatives I and II having a specific chemical structure were found toexhibit excellent plant growth regulatory effect far beyondexpectations.

In the present specification, those words which should be expressed initalics (or be underlined) may be expressed in non-italicized letters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a HPLC analysis chart of a reaction product of compound (4)and norepinephrine;

FIGS. 2A, 2B, and 2C show absorption spectra of compounds (5), (4), andnorepinephrine, respectively;

FIG. 3 shows flower bud formation promoting activity of compounds (4)and (5) on morning glory (Pharbitis nil);

FIG. 4 shows flower bud formation promoting activity of a reactionmixture of compound (4) and norepinephrine, and compound (5) on Lemnapaucicostata (duckweed);

FIG. 5 shows flower bud formation promoting activity of a reactionmixture of compound (4) and norepinephrine or an analogue ofnorepinephrine;

FIG. 6 shows the results of comparison regarding flower bud formationpromoting activity, on morning glory, of the ketol fatty acid derivative(9) of the invention and that of compound (4), etc.; and

FIG. 7 shows the results of study of flower bud formation promotingactivity, on morning glory, of the ketol fatty acid derivative (9) ofthe invention in relation to the administration concentration of thederivative.

BEST MODES FOR CARRYING OUT THE INVENTION

Modes for carrying out the invention will next be described.

A. Ketol Fatty Acid Derivatives I of the Invention

The ketol fatty acid derivatives I of the invention are α-ketolunsaturated fatty acid derivatives having a structure of formula (1).

As described above, R¹ in formula (1) is C₁₋₅ linear alkyl, and specificexamples thereof include methyl, ethyl, propyl, butyl and pentyl. In thepresent invention, R¹ is preferably methyl.

As described above, R² is hydrogen, hydroxyl, methyl, or ethyl. In thepresent invention, R² is preferably hydroxyl.

As described above, R³ is hydrogen or carboxyl. In the presentinvention, R³ is preferably hydrogen.

As described above, R⁴ is hydrogen, methyl, ethyl, isopropyl, acetyl, ortert-butyl. In the present invention, R⁴ is preferably methyl orhydrogen, with hydrogen being more preferred.

As described above, R⁵ is hydrogen, hydroxyl, methoxy, ethoxy, formyl,or hydroxymethyl. In the present invention, R⁵ is preferably hydroxyl orhydrogen, with hydroxyl being more preferred.

As described above, n is an integer from 3 to 15 inclusive. In thepresent invention, n is preferably an integer from 5 to 9 inclusive,with 7 being more preferred.

As described above, the carbon atoms at the A- and B-positions may be ineither (R)- or (S)-configuration. In the present invention, preferably,the carbon atom at the A-position is in (S)-configuration and that atthe B-position is in (R)-configuration.

Specific examples of the ketol fatty acid derivative I of the inventioninclude(9R)-11-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,(9S)-11-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,(9R)-11-{(2′R,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,

(9R)-11-{(8′S,10′S,11′R)-8′-hydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,(9R)-11-{(3′S,8′S,10′S,11′R)-3′-carboxy-8′-hydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,(9R)-11-{(8′S,10′S,11′R)-8′-hydroxy-4′-methyl-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,

(9S)-11-{(3′S,8′S,10′S,11′R)-3′-carboxy-8′-hydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,(9R)-11-{(3′S,10′S,11′R)-3′-carboxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,(9R)-11-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-heptenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoic acid,

(8R)-10-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-8-hydroxy-9-oxodecanoicacid,(11R)-13-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-11-hydroxy-12-oxotridecanoicacid,(9R)-11-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-hexenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid,

(11R)-13-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-hexenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-11-hydroxy-12-oxotridecanoicacid,(9R)-11-{(2′S,8′S,10′R,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid, and(9R)-11-{(2′S,8′S,10′R,11′S)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid.

Of the above-listed compounds,(9R)-11-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.01.5]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid is one of the most preferred species falling in the class of theketol fatty acid derivatives I of the invention.

The present invention is the first to have identified the ketol fattyacid derivatives I; in the past, the derivatives I had never beenidentified as either a natural product or a synthesized compound. Also,the derivatives I cannot be produced easily by use of chemical orenzymatic synthesis methods known per se.

The ketol fatty acid derivatives I of the invention exhibit a variety ofexcellent plant growth regulating activities, and therefore, they can beused as active components of plant growth regulators, which will bedescribed hereinbelow.

A characteristic feature of the ketol fatty acid derivatives I of theinvention is that they exhibit much stronger activities than do theaforementioned α-ketol unsaturated fatty acids of formula (2), whichwould be expected to exhibit similar levels of activities on plants.Specifically, the ketol fatty acid derivatives I of the inventionexhibit desired effects when used in an amount about 1/10,000 that ofα-ketol unsaturated fatty acids of formula (2).

B. Process of Producing the Ketol Fatty Acid Derivatives I of theInvention

As described above, the process of the invention is drawn to a processof preparing ketol fatty acid derivative I of the invention, and theprocess comprises the steps of mixing an α-ketol unsaturated fatty acidrepresented by formula (2) with a catecholamine represented by formula(3) (a typical example of which is norepinephrine) or a derivativethereof and incubating the resultant mixture under neutral to basicconditions, to thereby yield an α-ketol unsaturated fatty acidcatecholamine derivative. R¹ and n in formula (2) have the same meaningsas defined in connection with formula (1). Similarly, R² to R⁵ informula (3) have the same meanings as defined in connection with formula(1). As described above, R⁶ is hydrogen, linear or branched alkyl, oracyl. In the present invention, R⁶ is preferably hydrogen, C₁₋₃ linearor branched alkyl, or acyl, with hydrogen and methyl being morepreferred.

Some α-ketol unsaturated fatty acids (2), at the least, haveconventionally been known to be found in animals or plants asintermediates of fatty acid metabolites. Moreover, regarding, forexample, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (hereinaftermay be referred to as compound (4)), which is an α-ketol unsaturatedfatty acid (2) in which R¹ is methyl and n=7, this specific compound hasbeen known as an intermediate of an α-linolenic acid metabolite in wheat(Graveland, Lipids, 8, 606, 1973). Furthermore, the present inventorshave found that the aforementioned α-ketol unsaturated fatty acids (2),inclusive of compound (4), alone or in combination with norepinephrine(which is one form of catecholamine), exhibit flower bud formationpromoting effect for a broad range of plants (Japanese PatentApplication Laid-Open (kokai) No. 11-29410).

Examples of the α-ketol unsaturated fatty acids (2) include9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid,7-hydroxy-8-oxo-10(Z),13(Z)-octadecadienoic acid,9-hydroxy-10-oxo-12(Z),15(Z)-eicosadienoic acid,11-hydroxy-12-oxo-14(Z),17(Z)-eicosadienoic acid, and7-hydroxy-8-oxo-10(Z),13(Z)-nonadecadienoic acid.

Of these α-ketol unsaturated fatty acids (2), those contained in naturalproducts may be obtained by subjecting such natural products toextraction and purification. Alternatively, an α-ketol unsaturated fattyacid (2) of interest may be prepared by adding an enzyme such aslipoxygenase to α-linolenic acid, so as to cause reactions similar tothose in the fatty acid metabolism pathway in plants. Furtheralternatively, an α-ketol unsaturated fatty acid (2) of interest may beprepared through a chemical synthesis method known per se.

A detailed process for the production of α-ketol unsaturated fatty acid(2) is disclosed, for example, in Japanese Patent Application Laid-Open(kokai) No. 11-29410 or by Yokoyama et al. in Plant Cell Physiol., 41,110-113, 2000.

In the present invention, the catecholamine which is reacted withα-ketol unsaturated fatty acid (2); specifically, the catecholamine offormula (3) or a derivative thereof, is capable of forming anazatricyclo structure represented by formula (1) after having undergonethe reaction of the process of the present invention. Specific examplesthereof include norepinephrine, epinephrine, dopamine, normetanephrine,3-O-methyldopamine, and 4-O-methyldopamine.

The mentioned norepinephrine, epinephrine, and other catecholamines (3)may be those commercially available, may be obtained from naturalproducts through extraction and purification, or may be prepared by useof a chemical synthesis method known per se.

In this connection, norepinephrine is well known for its role as aneurotransmitter of the sympathetic nervous system in animals, but inaddition to being present in animals, this substance is also known to bepresent in plants. For example, norepinephrine has been found in a cellsuspension of Lemna paucicostata (Takimoto et al., Plant Cell Physiol.,30, 1017-1021, 1991). However, its role has not been completelyelucidated.

According to the process of the present invention, a desired derivativeof the invention can be obtained by subjecting α-ketol unsaturated fattyacid (2) and catecholamine (3) to incubation under neutral to basicconditions.

As used herein, the expression “neutral to basic conditions” encompassesa broad range of pH values, which would generally occur to those havingordinary skill in the art. Specifically, pH values of not lower than 6.0or thereabouts are intended, with weakly basic conditions of around pH 8to 9 being more preferred.

No particular limitation is imposed on the incubation time. Preferably,the incubation time is determined as 1 to 200 hours. Similarly, noparticular limitation is imposed on the incubation temperature.Preferably, the incubation temperature is determined as 4 to 40° C.

No particular limitation is imposed on the mixing ratio of α-ketolunsaturated fatty acid (2) and catecholamine (3) which are to beincubated. Preferably, the mixing ratio by weight is 1:10 to 10:1, with1:1 being most preferred.

Preferably, the reaction medium is water. However, the reaction mediummay be a hydrous solvent having a water content of at least 80 mass %.Examples of other solvents which may co-exist in water include methanol,ethanol, and acetone.

Because ketol fatty acid derivative I of the invention exhibits anabsorption peak in the vicinity of 300 nm, a UV detector may be employedto detect the derivative I contained in the reaction mixture resultingfrom an incubation procedure performed in accordance with the process ofthe invention. After detection, isolation means, such as HPLC, for acertain specified substance may be used, whereby the ketol fatty acidderivative I can be taken up from the reaction mixture through isolationand purification.

Alternatively, isolation and purification of the ketol fatty acidderivative I from the reaction mixture may be performed by use of amethod known per se. For example, extraction with a solvent or any of avariety of chromatography techniques may be employed.

C. Ketol Fatty Acid Derivatives II of the Invention

The ketol fatty acid derivatives II of the invention are α-ketolunsaturated fatty acid amide derivatives having a structure of formula(6).

As described above, R⁷ in formula (6) is C₁₋₁₇ alkyl or C₂₋₁₇unsaturated hydrocarbyl having 1 to 5 double bonds (wherein the numberof double bonds does not exceed the total number of carbon-carbonbonds).

Of the C₁₋₁₇ alkyl groups of R⁷, examples of linear alkyl groups includemethyl, ethyl, propyl, butyl, hexyl, octyl, decyl, dodecyl, tetradecyl,and pentadecyl. Similarly, examples of branched alkyl groups include2-methyldecyl, 4-ethylhexyl, 10-methyldodecyl, 3,5-diethyldodecyl,3,6,9-trimethyldecyl, and 2,2-isopropyl-3-methybutyl.

Also, of the C₂₋₁₇ unsaturated hydrocarbyl groups having 1 to 5 doublebonds (wherein the number of double bonds does not exceed the totalnumber of carbon-carbon bonds) of R⁷, examples of linear unsaturatedhydrocarbyl groups include 2-hexenyl, 2-pentenyl, 4-octenyl,2,5-heptadienyl, 3,5-octadienyl, 3,6,11-dodecatrienyl,1,3,6,8-hexadecatetraenyl, and 3,5,8,10,12-heptadecapentaenyl.Similarly, examples of branched unsaturated hydrocarbyl groups include3-ethyl-2-octenyl, 4-methyl-3-hexenyl, 7-methy-2,5-decadienyl,2-ethyl-5,7-dodecadienyl, and6-(1-propenyl)-8-isopropyl-1,4,9-decatrienyl.

Preferably, R⁷ in the ketol fatty acid derivative II of the invention isan alkenyl group of the following formula:R¹¹—HC═CH—CH₂—wherein R¹¹ is C₁₋₇ linear alkyl, and preferably ethyl, which has twocarbon atoms.

As described above, R⁸ in formula (6) represents hydrogen or a sidechain of α-amino acid. In the case where R⁸ is a side chain of α-aminoacid, no particular limitation is imposed on the species of the aminoacid, and examples thereof include glycine, alanine, leucine,isoleucine, tryptophane, tyrosine, glutamine, glutamic acid, asparagine,aspartic acid, lysine, arginine, histidine, cysteine, methionine,serine, phenylalanine, valine, proline, and threonine, regardless of theform L-, D-, or racemic. Of the listed α-amino acids, preferred areL-form neutral amino acids, specifically, L-alanine, L-valine,L-leucine, L-isoleucine, L-phenylalanine, etc., with L-isoleucine beingparticularly preferred.

As described above, R⁹ in formula (6) is an α-ketolalkylene group; i.e.,a divalent group represented by formula (7) or (8) shown below, with thecase of formula (7) being preferred, wherein the carbon atom on theamide bonding side of R⁹ is bonded to a hydroxyl group.

As described above, R¹⁰ in formula (6) is a divalent linear hydrocarbylgroup of the following formula:—(CH₂)_(L)—(HC═CH)_(m)—(CH₂)_(k)—wherein L, m, and k are integers, with m being 0 or 1 and L+k being 1 to15 inclusive. Preferably, m is 0 and L+k is 3 to 10 inclusive, and morepreferably, m is 0 and L+k is 7.

Preferably, double bonds contained in the ketol fatty acid derivative IIof the invention are of the cis-form. Specifically, the following doublebonds:

the double bond appearing in R⁷ of R¹¹—HC═CH—CH₂—,

the double bond appearing in R¹⁰ when m=1, and

the double bond appearing in the moiety R⁷—HC═CH—CH₂— of the ketol fattyacid derivative II of the invention, are preferably of the cis-form.

A ketol fatty acid derivative II of the invention contain a plurality ofasymmetric carbon atoms, and any of the stereoisomers attributed tothose asymmetric carbon atoms falls within the definition of the ketolfatty acid derivative II of the invention. Thus, the ketol fatty acidderivatives II of the invention may be optical isomers derived fromoptical resolution or may be mixtures of such isomers.

Specific examples of the ketol fatty acid derivative II of the inventioninclude N-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-L-isoleucine,N-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-L-serine,N-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-L-asparagine,N-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-L-leucine,N-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-L-histidine,N-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-glycine,N-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-D-isoleucine,N-[9-hydroxy-10-oxo-12(Z)-octadecaenoyl]-L-isoleucine,N-[13-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoyl]-L-valine,N-[13-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoyl]-D-alanine,N-[9-hydroxy-10-oxo-12(Z),15(Z)-eicosadienoyl]-L-isoleucine,

N-[9-hydroxy-10-oxo-12(Z),15-hexadecadienoyl]-L-isoleucine,N-[9-hydroxy-10-oxo-6(Z),12(Z)-octadecadienoyl]-L-leucine,N-[9-hydroxy-10-oxo-12(E),15(E)-octadecadienoyl]-L-isoleucine,N-[9-hydroxy-10-oxo-12(E),15(E)-octadecadienoyl]-D-leucine,N-[9-hydroxy-10-oxo-6(Z),12(Z),15(Z)-octadecatrienoyl]-L-isoleucine,N-[9-hydroxy-17-methyl-10-oxo-12(Z),15(Z)-octadecadienoyl]-L-isoleucine,N-[9-hydroxy-17-methyl-10-oxo-12(Z),15(Z)-octadecadienoyl]-D-asparticacid, andN-[15,17-dimethyl-9-hydroxy-1′-oxo-12(Z)-octadecaenoyl]-L-isoleucine.

Of these listed ketol fatty acid derivatives II of the invention,N-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-L-isoleucine is one ofbest preferred, because it satisfies all the above-mentioned preferredconditions.

The ketol fatty acid derivatives II of the invention are endowed with avariety of excellent plant growth regulating effects, and therefore, canbe used as an active ingredient of plant growth regulators, which areemployed for activating plants, as described hereinbelow.

A characteristic feature of the ketol fatty acid derivatives II of theinvention is that they exhibit much stronger activities than do α-ketolunsaturated fatty acids disclosed in Japanese Patent ApplicationLaid-Open (kokai) No. 11-29410, which are represented by formula (6)whose amino acid moiety (—NH—CHR⁸—COOH) is replaced by a hydroxyl group(—OH). In other words, the ketol fatty acid derivatives II of theinvention exhibit much stronger activities than do α-ketol unsaturatedfatty acids (2), which would be expected to exhibit similar levels ofactivities on plants.

It should be noted that the ketol fatty acid derivatives II of theinvention had never been identified as either a natural product or asynthesized compound.

The ketol fatty acid derivatives II of the invention can be produced bya process which accords with the specific chemical structure of thederivatives of interest. For example, the ketol fatty acid derivativesII can be produced by causing the 1-position carboxyl of α-ketolunsaturated fatty acid (2) to be bonded to the amino group of α-aminoacid.

Some α-ketol unsaturated fatty acids (2), at the least, which may serveas starting substances in the production of the ketol fatty acidderivatives II of the invention, have conventionally been known to befound in animals or plants as intermediates of fatty acid metabolites.Moreover, the present inventors has already found that theabove-mentioned α-ketol unsaturated fatty acids (2), such as9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid, exhibit flowe budformation promoting effect (Japanese Patent Application Laid-Open(kokai) No. 11-29410).

Examples of the α-ketol unsaturated fatty acids (2) include9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid,7-hydroxy-8-oxo-10(Z),13(Z)-octadecadienoic acid,9-hydroxy-10-oxo-12(Z),15(Z)-eicosadienoic acid,11-hydroxy-12-oxo-14(Z),17(Z)-eicosadienoic acid, and7-hydroxy-8-oxo-10(Z),13(Z)-nonadecadienoic acid.

Of these α-ketol unsaturated fatty acids (2), those contained in naturalproducts may be obtained by subjecting such natural products toextraction and purification. Alternatively, an α-ketol unsaturated fattyacid (2) of interest may be prepared by adding an enzyme such aslipoxygenase to α-linolenic acid, so as to cause reactions similar tothose in the fatty acid metabolism pathway in plants. Furtheralternatively, an α-ketol unsaturated fatty acid (2) of interest may beprepared through a chemical synthesis method known per se.

A detailed process for the production of α-ketol unsaturated fatty acid(2) is disclosed, for example, in Japanese Patent Application Laid-Open(kokai) No. 11-29410 or by Yokoyama et al. in Plant Cell Physiol., 41,110-113, 2000.

Bonding between the 1-position carboxyl of α-ketol unsaturated fattyacid (2) and the amino group of α-amino acid may be achieved through atechnique known per se. For example, as is described in literature suchas “Lectures on Experimental Chemistry 22, Organic Synthesis IV. Acids,Amino Acids, and Peptides” pp. 259-271 (edited by the Chemical Societyof Japan, published by Maruzen Co., Ltd., 1992) and “Fundamentals andExperiments of Peptide Syntheses” authored by IZUMIYA, Nobuo, et al.,pp. 89-125 (Maruzen Co., Ltd., 1985), there may be employed thecondensation method, the active esterification method, the azide method,or the mixed acid anhydride method.

When the condensation method is employed, example condensing agentswhich may be used include dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIPC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC) or a hydrochloricsalt thereof, benzotriazol-1-yl-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP), and diphenylphosphorylazide (DPPA). Ifnecessary, 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HONSu),3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt), or the likecompound may be employed as an additive.

For example, a target ketol fatty acid derivative II may be obtainedthrough the following process. Firstly, α-ketol unsaturated fatty acid(2) and α-amino acid are dissolved in a solvent such as DMF. Theresultant solution is allowed to react in the presence of a condensingagent or a combination of a condensing agent and an additive.Subsequently, the reaction mixture is subjected to further treatmentssuch as concentration and purification.

When the active esterification method is employed, there may be usedp-nitrophenol, N-hydroxysuccinimide, pentafluorophenol, etc. Briefly, ap-nitrophenyl ester (ONp), an N-hydroxysuccinimide ester (ONSu), apentafluorophenyl ester (OPfp), or a similar ester of α-ketolunsaturated fatty acid is formed, and the ester is subjected to acondensation reaction with an amino acid of interest, whereby a targetketol fatty acid derivative II is obtained.

When the azide method is performed, hydrazine, anhydrous hydrazine, or asimilar substance may be employed. Through the process, a hydrizide ofα-ketol unsaturated fatty acid is obtained. Subsequently, by use of, forexample, butyl nitrite or tert-butyl nitrite, an azide is obtained,which is subjected to a condensation reaction with an amino acid ofinterest, whereby a target ketol fatty acid derivative II is obtained.

When the mixed acid anhydride method is performed, isobutyloxycarbonylchloride, diethylacetyl chloride, trimethylacetyl chloride, or a similarsubstance may be employed. Through the process, an anhydride of α-ketolunsaturated fatty acid is formed, which is subsequently subjected to acondensation reaction with an amino acid of interest, whereby a targetketol fatty acid derivative II is obtained.

When any of the above reactions for preparing the ketol fatty acidderivative II of the invention is performed, according to needs, aprotective group may be introduced and ultimately removed.

Thus, the ketol fatty acid derivatives II of the invention having avariety of excellent plant growth regulating effects can be prepared.

D. The Plant Growth Regulator of the Invention

When applied to a plant, the plant growth regulator of the invention canregulate the growth of the plant.

By the expression “plant growth regulation” or “regulation of plantgrowth,” the following effects, among other effects, are contemplated.

(a) Flower Bud Formation Promoting Effect

Administration of the plant growth regulator of the invention to a plantin need thereof promotes formation of flower buds of the plant. That is,by the application of the plant growth regulator of the invention,formation of flower buds—which precede flowering of the plant—can beinduced.

In this sense, the present invention provides an agent exerting a morespecified effect of “promotion of flower bud formation of a plant”(flower bud formation promoter).

When the plant growth regulator of the invention is applied to a plantin expectations of promoting flower bud formation, no particularlimitation is imposed on the timing of administration, so long as theadministration is performed before the formation of flower buds.Preferably, the regulator is administered to a plant while the plantundergoes other treatment suitable for the plant. For example, in thecase of a short-day plant such as morning glory (Pharbitis nil),preferably, administration of the plant growth regulator of theinvention is performed in combination with a predetermined dark periodtreatment.

(b) Plant Activating Effect

Through administration of the plant growth regulator of the invention toa plant, the life-related activities of the plant can be activated.

Specifically, examples of such plant activators include plant growthpromoters, anti-aging agents, dormancy preventive agents, andanti-stress agents.

(b)-1: Plant Growth Promoting Effect

When the plant growth regulator of the invention is used as a plantactivator, it accelerates the growth rate of the plant to therebyimprove harvest efficiency, etc.; i.e., the plant growth regulator ofthe invention may be used as a plant growth promoter. (As describedhereinabove, attainable effects in this case include enlargement instems and leaves, growth promotion of tubers and root tubers, promotionof fruit bearing, and fruit growth promotion). In this sense, thepresent invention provides an agent exerting a more specified effect of“promotion of plant growth” (plant growth promoter).

When the plant growth regulator of the invention is applied to a plantfor the purpose of activating the plant, a notable effect occurs; growthof a plant can be accelerated in an early stage after germination, whichpreviously had been difficult to attain by use of a fertilizer.

Therefore, when the plant growth regulator of the invention is used as aplant growth promoter, it is recommended that the regulator beadministered at the time of seeding or during an early stage of growthof a plant after germination.

That is, simple administration, by spraying or any other means, of theplant growth regulator of the invention during an early stage of growthof a plant after germination persistently promotes the growth of theplant. Also, even when the plant growth regulator of the invention isused excessively, plant growth disorder is observed only in rare cases,as contrasted with case of excessive application of fertilizers, andtherefore, the plant growth regulator can be used without paying strictattention to the amount applied.

In the fields of horticulture or agriculture, instead of seeds, whosehandling is troublesome after delivery thereof, distribution in the formof seedlings is now becoming mainstream. In particular, most amateurflower gardeners purchase seedlings rather than seeds from ornamentalflower businesses. Thus, if the plant growth regulator is used beforetrading or distribution of the seedlings, well-grown seedlings can beput on the market.

The above-described properties of the plant growth regulator of theinvention are advantageously used for attaining increased yields ofleafy vegetables, such as spinach, lettuce, and cabbage.

(b)-2: Anti-Aging Effect

The plant growth regulator of the invention may be used as a plantactivator, which is an agent capable of activating a plant by retardingaging. Specifically, the regulator of the invention can prolong theflowering period, whereby people can enjoy flowers for a longer period,or whereby the pollination period can be extended. The present inventionalso provides “flowering period extenders” on the basis of such anindividual effect; i.e., an effect of extending the flowering period. Inaddition, administration of the plant growth regulator of the inventioncan increase the number of flowers per plant.

When the plant growth regulator of the invention is employed as a plantactivator in expectation of prolonging the flowering period, theregulator can be administered to a plant at any time, including theflowering period of the plant. Specifically, administration may beperformed while seeds are soaked in water or after germination.

As is sometimes experienced with the case of annual plants, when a plantbecomes very weak and withers, administration of the plant growthregulator of the invention to such a plant can delay withering (oraging) of the plant.

Thus, the plant growth regulator of the invention exerts the specificeffects of “flowering period prolonging effect” and “life-extendingeffect” on plants; in other words, a “plant aging retarding effect” onplants, to thereby activate the plants.

Thus, the present invention also provides an agent exerting a specific“plant aging retarding effect” among other plant activating effects(plant aging retardant).

(b)-3: Dormancy Inhibiting Effect

Through administration of the plant growth regulator of the invention toa plant, the plant can be prevented from falling into a dormant state,whereby the plant can be activated. Specifically, when the plant growthregulator of the invention is used as a plant activator, the “dormancyperiod,” during which growth of a plant is stopped for a certain periodbecause of deterioration of the growth environment, can be shortened orterminated.

In this sense, the present invention also provides an agent exerting aspecific “plant dormancy inhibiting effect” (plant dormancy inhibitor).

When the plant growth regulator of the invention is used as a plantactivator in expectation of inhibiting dormancy of a plant,administration thereof is preferably performed in an early stage aftergermination of the plant, to thereby successfully prevent the plant fromfalling dormant. In this connection, when the plant growth regulator isadministered to a plant which has already fallen into dormancy, theplant can be revived.

(b)-4: Anti-Stress Effect

Administration of the plant growth regulator of the invention to a plantcan impart stress resistance to the plant, thereby activating the plant.Plants are under a variety of stresses, including dryness, hightemperature, low temperature, osmotic pressure, and so on. Thus, by useof the plant growth regulator of the invention, stresses imposed onplants by climate changes or by germination-inducing treatment—which maylower the harvest yield of cultivated plants—can be mitigated, wherebythe plants can be activated.

In this sense, the present invention also provides an agent that exertsa specific “stress mitigating effect” on plants (plant stresssuppressor).

When the plant growth regulator of the invention is used as a plantactivator in expectation of suppressing plant stress, administrationthereof is preferably performed when germination of plant seeds isinduced, or after germination, to thereby successfully impart to theplant resistance against stresses.

No particular limitation is imposed on the upper limit of theadministration amount of the active agent of the plant growth regulatorof the invention, ketol fatty acid derivative I or II. That is, evenwhen the ketol fatty acid derivative I or II, as contained in the plantgrowth regulator of the invention, is administered to a plant in largeamounts, negative effects on the plant, such as growth disturbance, arerarely observed. Thus, as compared with the case of conventional planthormonal agents, which produce considerable negative effects ifadministered in excessive amounts, and thus require careful control ofthe amount to be administered, the plant growth regulator of theinvention is understood to be a remarkably excellent agent.

The lower limit of the administration amount of the ketol fatty acidderivative I or II may differ depending on the identity or size ofindividual plants. As a general tip, the lower limit for ketol fattyacid derivative I would be 0.1 nM, and that for ketol fatty acidderivative II would be 0.01 μM, for a single plant per administration.

The amount of ketol fatty acid derivative I or II to be incorporatedinto the plant growth regulator of the invention can be determined inaccordance with the manner of use, the identity of the plant to whichthe regulator is applied, the specific product form of the regulator,and other factors. The ketol fatty acid derivative I or II per se may beused as the plant growth regulator of the invention. In consideration ofthe typical dose of the ketol fatty acid derivative I or II, when ketolfatty acid derivative I is incorporated into the regulator, the amountof the derivative I is preferably about 0.1 ppb to about 1 ppm, morepreferably about 1 ppb to about 100 ppb, on the basis of the entirety ofthe regulator. When ketol fatty acid derivative II is incorporated intothe regulator, the amount of the derivative II is preferably about 10ppb to about 100 ppm, more preferably about 100 ppb to about 10 ppm, onthe basis of the entirety of the regulator.

Examples of the product form of the plant growth regulator of theinvention include solutions, solid agents, powders, and emulsions. Inaccordance with the product form, the plant growth regulator of theinvention may appropriately contain known pharmaceutically acceptablecarrier components and auxiliary agents for drug production, so long asthey do not impede the intended effect of the present invention; i.e.,plant growth promoting effect. When the plant growth regulator of theinvention assumes the form of powders or solid agents, for example, thefollowing carrier components may be incorporated: inorganic substancessuch as talc, clay, vermiculite, diatomaceous earth, kaolin, calciumcarbonate, calcium hydroxide, terra alba, and silica gel; and solidcarriers such as flour and starch. When the plant growth regulator ofthe invention assumes the form of solutions, for example, the followingcarrier components may be incorporated: liquid carriers including water;aromatic hydrocarbons such as xylene; alcohols such as ethanol andethylene glycols; ketones such as acetone; ethers such as dioxane andtetrahydrofuran; dimethylformamide; dimethyl sulfoxide; andacetonitrile. Examples of the auxiliary agents for drug production whichmay be incorporated include anionic surfactants such as alkyl sulfates,alkyl sulfonates, alkyl aryl sulfonates, dialkyl sulfosuccinates;cationic surfactants such as salts of higher aliphatic amines; nonionicsurfactants such as polyoxyethylene glycol alkyl ethers, polyoxyethyleneglycol acyl esters, polyoxyethylene polyalcohol acyl esters, andcellulose derivatives; thickeners such as gelatin, casein, gum arabi;extenders; and binders.

If desired, the plant growth regulator of the invention may furthercontain typical plant growth controlling agents, benzoic acid, nicotinicacid, nicotinamide, and pipecolic acid, so long as they do not impedethe intended effects of the present invention.

The plant growth regulator of the invention may be applied to variousplants in a manner in accordance with the product form of the regulator.For example, the plant growth regulator of the invention may be sprayed,dropped, or applied, in the form of solution or emulsion, to the pointof growth of a plant, to a portion of the plant, such as stem or leaf,or to the entirety of the plant; or may be absorbed, in the form ofsolid agent or powder, in the root of the plant via earth.Alternatively, when the plant growth regulator of the invention is usedfor promoting growth of a water plant such as duckweed, the regulatormay be absorbed in the root of the water plant, or the regulator in theform of solid agent may be gradually dissolve in the water.

The frequency of application of the plant growth regulator of theinvention to a plant varies with the type of the plant or the purpose ofapplication. Basically, desired effects can be obtained through merely asingle application. When the regulator is applied several times,application is preferably performed at an interval of one week or more.

No particular limitation is imposed on the type of plants to which theplant growth regulator of the invention can be applied, and theregulator is effective for angiosperms (dicotyledons andmonocotyledons), fungi, lichens, bryophytes, ferns, and gymnosperms.

Examples of dicotylendos of angiosperms include Convolvulaceae such asConvolvulus (C. nil L.; Pharbitis nil), Calystegia (C. japonica, C.hederacea, and C. soldanella), Ipomoea (I. pes-caprae, and I. batatas),and Cuscuta (C. japonica, and C. australis), Caryophyllaceae such asDianthus, Stellaria, Minuartia, Cerastium, Sagina, Arenaria, Moehringia,Pseudostellaria, Honkenya, Spergula, Spergularia, Silene, Lychnis,Melandryum, and Cucubalus, and furthermore, Casuarinaceae, Saururaceae,Piperaceae, Chloranthaceae, Salicaceae, Myricaceae, Juglandaceae,Betulaceae, Fagaceae, Ulmaceae, Moraceae, Urticaceae, Podostemaceae,Proteaceae, Olacaceae, Santalaceae, Loranthaceae, Aristolochiaceae,Mitrastemonaceae, Balanophoraceae, Polygonaceae, Chenopodiaceae,Amaranthaceae, Nyctaginaceae, Theligonaceae, Phytolaccaceae, Aizoaceae,Portulacaceae, Magnoliaceae, Trochodendraceae, Cercidiphyllaceae,Nymphaeaceae, Ceratophyllaceae, Ranunculaceae, Lardizabalaceae,Berberidaceae, Menispermaceae, Calycanthaceae, Lauraceae, Papaveraceae,Capparaceae, Brassicaceae (Crusiferae), Droseraceae, Nepenthaceae,Crassulaceae, Saxifragaceae, Pittosporaceae, Hamamelidaceae,Platanaceae, Rosaceae, Fabaceae (Leguminosae), Oxalidaceae, Geraniaceae,Linaceae, Zygophyllaceae, Rutaceae, Simaroubaceae, Meliaceae,Polygolaceae, Euphorbiaceae, Callitrichaceae, Empetraceae, Coriariaceae,Anacardiaceae, Aquifoliaceae, Celastraceae, Staphyleaceae, Icacinaceae,Aceraceae, Hippocastanaceae, Sapindaceae, Sabiaceae, Balsaminaceae,Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvales,Sterculiaceae, Actinidiaceae, Theaceae, Clusiaceae (Guttiferae),Elatinaceae, Tamaricaceae, Violaceae, Flacourtiaceae, Stachyuraceae,Possifloraceae, Begoniaceae, Cactaceae, Thymelaeaceae, Elaeagnaceae,Lythraceae, Punicaceae, Rhizophoraceae, Alangiaceae, Melastomataceae,Trapaceae, Onagraceae, Haloragaceae, Hippuridaceae, Araliaceae, Apiaceae(Umbelliferae), Cornaceae, Diapensiaceae, Clethraceae, Pyrolaceae,Ericaceae, Myrsinaceae, Primulaceae, Plumbaginaceae, Ebenaceae,Symplocaceae, Styracaceae, Oleaceae, Buddlejaceae, Gentianaceae,Apocynaceae, Asclepiadaceae, Polemoniaceae, Boraginaceae, Verbenaceae,Lamiaceae (Labiatae), Solanaceae (Solanum, Lycoperisicon, etc.),Scrophulariaceae, Bignoniaceae, Pedaliaceae, Orobanchaceae,Gesneriaceae, Lentibulariaceae, Acanthaceae, Myoporaceae, Phrymaceae,Plantaginaceae, Rubiaceae, Caprifoliaceae, Adoxaceae, Valerianaceae,Dipsacaceae, Cucurbitaceae, Campanulaceae, and Asteraceae (Compositae).

Examples of monocotyledons include Lemnaceae such as Spirodela (S.polyrhiza), and Lemna (L. paucicostata, and L. trisulcata), Orchidaceaesuch as Cattleya, Cymbidium, Dendrobium, Phalaenopsis, Vanda,Paphiopedilum, and Oncidium, Typhaceae, Sparganiaceae, Potamogetonaceae,Najadaceae, Scheuchzeriaceae, Alismataceae, Hydrocharitaceae,Triuridaceae, Poaceae (Gramineae) (Oryza, Hordeum, Triticum, Secale,Zea, etc.), Cyperaceae, Arecaceae (Palmae), Araceae, Eriocaulaceae,Commelinaceae, Pontederiaceae, Juncaceae, Stemonaceae, Liliaceae(Asparagus, etc.), Amaryllidaceae, Dioscoreaceae, Iridaceae, Musaceae,Zingiberaceae, Cannaceae, and Burmanniaceae.

EXAMPLES

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the technical scopeof the invention thereto.

Example 1 Production, Isolation/Purification, and Structural Analysis of(9R)-11-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicAcid (Hereinafter may be Referred to as Compound (5))

Compound (4) (100 mg) was dissolved in water (500 mL), and(R)-(−)-norepinephrine hydrogentartrate monohydrate (111.3 mg, Wako PureChemical Industries, Ltd.) and 1M Tris buffer (pH 8.0, 16.1 mL) wereadded thereto, followed by incubation at 25° C. for 102 hours. Theresultant solution was concentrated under reduced pressure, and theconcentrate was subjected to fractionation by means of silica gel columnchromatography (Wakogel C-200). By use of a mixture of chloroform,methanol, and water (10:3:1) as an eluting agent, fractionation wasfurther carried out by means of HPLC (column: Capcellpak C-18 UG120,4.6×250 mm, solvent: 25% acetonitrile+0.1% trifluoroacetic acid, flowrate: 1 mL/min, wavelength: 300 nm). The results of HPLC analysis areshown in FIG. 1. A fraction corresponding to the highest peak (retentiontime: 26.8 min) was applied to a column having a larger size (CapcellpakC-18 UG120, 10×250 mm) for fractionation. The resultant fraction wasfreeze-dried, to thereby yield 4.5 mg of compound (5) as yellowcrystals.

The following instruments were employed for structural analyses ofcompound (5): A SEPA-300 digital polarimeter (Horiba Ltd.) formeasurement of angle of rotation; a UV-2200 spectrophotometer (ShimadzuCorporation) for measurement of absorption spectrum; an FT/IR-5300(JASCO Corporation) for measurement of IR spectrum; a MAT95Q (FinniganMAT) for measurement of high-resolution FAB-MS; and an ECP-400 (JEOL)for measurement of ¹H-NMR and ¹³C-NMR. The absorption spectrum measuredwithin the range of 200 nm to 600 nm is shown in FIG. 2 (FIG. 2A, FIG.2B, and FIG. 2C show absorption spectra of compound (5), compound (4),and norepinephrine, respectively, wherein the X axes representwavelength (nm), and the Y axes represent absorbance values).

Angle of rotation: [α]_(D) ²⁵+40.5° (c=0.2, MeOH)

High-resolution FAB-MS: 494.27789 (M+H)⁺

UV λ max(MeOH):292 nm (ε=12000), 205 nm (ε=3500)

IR (KBr, cm⁻¹):3400, 1695, 1685, 1630

¹H-NMR (400 MHz, CD₃OD): δ 1.03 (3H, t, J=7.3, 5″-H₃), 1.11-1.32 (8H,4,5,6,7-H₂), 1.22 (1H, m, 3-H), 1.59 (1H, t, J=12.0, 3-H), 1.44 (1H, m,11′-H), 1.48, 1.66 (both 1H, m, 8-H₂), 2.00, 2.10(both 1H, d, J=12.0,12′-H₂), 2.11, 2.65 (both 1H, m, 1″-H₂), 2.17 (2H, m, J=7.3, 4″-H₂),2.26 (2H, t-like, J=7.3, 2-H₂), 2.56 (1H, dd, J=7.8, 17.0, 11-H), 2.86(1H, dd, J=5.0, 17.0, 11-H), 3.43 (1H, d, J=13.0, 3′-H), 3.85 (1H, dd,J=3.4, 13.0, 3′-H), 3.98 (1H, dd, J=4.0, 7.7, 9-H), 4.29 (1H, d, J=3.4,2′-H), 4.58 (1H, m, 10′-H), 5.25 (1H, m, 2″-H), 5.36 (1H, m, 3″-H), 5.42(1H, s, 6′-H).

¹³C-NMR (100 MHz, CD₃OD):15.0 (C-5″), 22.7 (C-4″), 25.7 (C-1″), 26.3,26.4 (C-3,4), 31.0, 31.0, 31.0 (C-5,6,7), 33.6 (C-12′), 34.9 (C-8), 35.7(C-2), 41.5 (C-11′), 42.9 (C-11), 56.1 (C-3′), 59.5 (C-1′), 72.6(C-10′), 74.2 (C-2′), 79.0 (C-9), 95.0 (C-6′), 95.8 (C-8′), 131.0(C-2″), 134.2 (C-3″), 177.3 (C-5′), 178.5 (C-1), 189.0 (C-7′), 213.1(C-10).

Assignments of proton and carbon signals were determined throughmeasurement of two-dimensional NMR spectra, such as DIFCOSY, HMQC, andHOHAHA spectra. Comparison of the thus-obtained data with data ofcompound (4) (described in Japanese Patent Application Laid-Open (kokai)No. 11-29410 and Yokoyama et al., Plant Cell Physiol., 41, 110-113,2000) reveals that a signal corresponding to cis-olefin at the12-position of compound (4) had disappeared, suggesting addition of somecompound to compound (5) at the 12-position.

When compound (5) was acetylated by use of acetic anhydride/pyridine, atetraacetate derivative of compound (5) was obtained, proving thatcompound (5) has four active protons. By comparison of ¹H-NMR data ofthe tetraacetate derivative with those of compound (5), signalscorresponding to protons at the 9-, 2′-, and 3′-position of compound (5)were found to have shifted to a lower magnetic field side, whereas theproton signal at the 10′-position was not found to have shifted.Therefore, compound (5) is considered to have an ether-linkage at the10′-position. In addition, these results and detailed analyses of HMBCspectra indicate that compound (5) has a tricyclic structure. Moreover,when NOESY and GOESY spectra of compound (5) were measured, NOEs werefound to be observed, for example, between the 11′-position proton andthe 2′-, 3′-, and 10′-position protons; between the 10′-position protonand the 6′-position proton; and between the 12′-position axial protonand the 1″-position proton. As a result, 3-dimensional configuration ofthe cyclic moiety of compound (5) is clearly recognized.

In order to determine the absolute configuration of the 9-position ofcompound (5), first, absolute configuration of the 9-position ofcompound (4) which had been obtained through enzyme synthesis wasanalyzed. Methyl ester reductant of compound (4) was reacted with(R)-(+)- or (S)-(−)-2-methoxy-2-trifluoromethylphenylacetic acid (MTPA)in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride and 4-dimethylaminopyridine, whereby respective (R)- or(S)-MTPA ester was obtained. The MTPA esters were applied to themodified Mosher method (Ohtani et al., J. Am. Chem. Soc., 113,4092-4096, 1991). The results indicate that compound (4) obtainedthrough enzyme synthesis is a mixture of 70% (9R)- and 30% (9S)-opticalisomers.

Each of (9R)- and (9S)-optical isomers of compound (4) which had beenfractionated by use of a chiral column was reacted with norepinephrine.As a result, only (9R)-configuration of compound (4) was found to yieldcompound (5).

From the above-described data, the structure of compound (5) isdetermined as follows.

Example 2 Measurement of Flower Bud Formation Promoting Activity ofCompounds (4) and (5) in Morning Glory (Pharbitis nil)

Seeds (9 g) of morning glory (variety name: Murasaki) were treated withconcentrated sulfuric acid for 20 minutes, and then left to standovernight under running water. Subsequently, the treated seeds wereplaced on wet sea sand for 24 hours, hilum side up, to thereby formroots. The seeds having roots were planted in sea sand to a depth of 1.5cm to 2.0 cm, followed by culturing under continuous light (forapproximately 5 days).

The whole plants of the morning glory which had put forth leaves throughthe culture were transferred to a culture solution [KNO₃ (250 mg),NH₄NO₃ (250 mg), KH₂PO₄ (250 mg), MgSO₄.7H₂O (250 mg), MnSO₄.4H₂O (1mg), Fe-citrate n-hydrate (6 mg), H₃BO₃ (2 mg), CuSO₄.5H₂O (0.1 mg),ZnSO₄.7H₂O (0.2 mg), Na₂MoO₄.2H₂O (0.2 mg), Ca(H₂PO₄)₂.2H₂O (250mg)/distilled water (1,000 mL)].

Compound (4) or compound (5) was sprayed onto the cotyledons of theabove morning glory plants at a spray volume of 2 mL per eight plants.The morning glory plants were subjected to treatment in the dark (14hours), then raised under the application of continuous light at 26° C.for 14 days. On day 14, the number of flower buds of the morning gloryplants was counted.

The results are shown in FIG. 3. As a control, distilled water wassprayed onto a group of plants. Compound (4) was sprayed at aconcentration of 100 μM, whereas compound (5) was sprayed atconcentrations of 0.001 μM to 100 μM.

Compound (5), having a significantly low concentration as compared withcompound (4), exhibited flower bud formation promoting activity and 0.01μM compound (5) exhibited the same level of the activity as 100 μMcompound (4).

Example 3 Measurement of Flower Bud Formation Promoting Activity ofCompound (5) in Lemna paucicostata (Duckweed)

The flower bud formation promoting effect of compound (5) was measuredon the basis of percent flower generation (%, number of thalluses inwhich flower bud formation is confirmed/number of all thalluses×100) byuse of duckweed P151 plants as model plants.

Compound (4) (0.155 mg) was dissolved in water (0.15 mL), and 10 mM(R)-(−)-norepinephrine hydrogentartrate monohydrate solution (50 μL) and0.5M Tris buffer (pH 8.0, 25 μL) were added thereto, followed byincubation at 25° C. for 6 hours.

Subsequently, a predetermined volume or amount of the incubated solutionor compound (5) prepared in the above Example 1 was added to assaymedium (10 mL, no sucrose-containing 1/10 E medium+1 μM benzyladenine)in a flask.

Onto each of the above assay media, a colony of the P151 plants wasplanted, followed by incubation at 24-25° C. for 7 days with continuousirradiation by use of a daylight-type fluorescent lamp (Hitachi FL20SSD, approximately 5 W/m²), to thereby determine the percent flowergeneration (FIG. 4).

Compound (5) was found to have flower bud formation promoting activity,although the level of the activity of compound (5) was slightly lowerthan that of a reaction mixture of compound (4) and norepinephrine. Thisis the first time that a single substance has been found to induceformation of flower bud in duckweed.

Example 4 Measurement of Flower Bud Formation Promoting Activity ofReaction Mixture of Compound (4) and Norepinephrine or Analogues Thereofin Lemna paucicostata

The flower bud formation promoting effect of a reaction mixture ofcompound (4) and norepinephrine (L-norepinephrine) or analogues thereofwas measured on the basis of percent flower generation (%, number ofthalluses in which flower bud formation is confirmed/number of allthalluses×100) by use of duckweed P151 plants as model plants.

The employed analogues of norepinephrine are L-epinephrine, dopamine,DL-normetanephrine, 3-O-methyldopamine, 4-O-methyldopamine, homovanillicacid, and vanilloylmandelic acid.

Compound (4) (0.155 mg) was dissolved in water (0.092 mL), and 10 mM(R)-(−)-norepinephrine hydrogentartrate monohydrate solution (50 μL) ora solution (50 μL) containing each of the analogues and 0.5M Tris buffer(pH 8.0, 25 μL) were added thereto, followed by incubation at 25° C. for24 hours.

Subsequently, the above-incubated solution (10 μL) or a 10-fold dilutedsolution thereof (10 μL) was added to assay medium (10 mL, nosucrose-containing 1/10 E medium+1 μM benzyladenine) in a flask.

Onto each of the above assay media, a colony of the P151 plants wasplanted, followed by incubation at 24-25° C. for 7 days with continuousirradiation by use of a daylight-type fluorescent lamp (Hitachi FL20SSD, approximately 10 W/m²), to thereby determine the percent flowergeneration (FIG. 5).

In addition to a reaction mixture of compound (4) and norepinephrine, areaction mixture of compound (4) and dopamine or 4-O-methyldopamine wasfound to exhibit high flower bud formation promoting activity. Moreover,a reaction mixture of compound (4) and L-epinephrine,DL-normetanephrine, or 3-O-methyldopamine was found to exhibit a certainlevel of flower bud formation promoting activity. In contrast, areaction mixture of compound (4) and homovanillic acid orvanilloylmandelic acid was found to exhibit no appreciable flower budformation promoting activity.

Example 5 Synthesis ofN-[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoyl]-L-isoleucine(Hereinafter may be Referred to as Ketol Fatty Acid Derivative (9) ofthe Invention)

9-Hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (compound (4),produced through the “enzyme method” described in paragraph Nos. 0046 to0065 of Japanese Patent Application Laid-Open (kokai) No. 11-29410, 14.7mg, 0.0474 mmol) was dissolved in a mixture (5 mL) of DMF and H₂O (9:1).WSC (8.17 mg, 0.9 eq.), HOBt (6.52 mg, 0.9 eq.), and N-methylmorpholine(52.1 μL, 10 eq.) were added thereto, followed by stirring at roomtemperature for 20 minutes. L-isoleucine lithium salt (13.0 mg, 2 eq.)was added to the stirred solution, followed by a further stirring atroom temperature for 2 hours. The resultant solution was subjected toisolation and purification by means of HPLC (Capcellpak C18 UG 120 A, 5μm, 10 mm×250 mm: solvent [a mixture of CH₃CN (containing 0.085% TFA)and H₂O (containing 0.1% TFA) (1:1)]), to thereby yield 3.0 mg of ketolfatty acid derivative (9) of the invention.

The structure of the ketol fatty acid derivative (9) of the inventionwas analyzed by use of ¹H- and ¹³C-NMR.

¹H-NMR (400 MHz, CDCl₃): 0.92 (3H, t, J=7.3, 5′-H₃), 0.96 (3H, t, J=7.3,18-H₃), 0.98 (3H, d, J=7.3, 6′-H₃), 1.18, 1.54 (2H, both m, 4H′-H₂),1.20-1.50 (all 8H, m, 4,5,6,7-H₂), 1.52, 1.93 (2H, both m, 8-H₂), 1.60(2H, m, 3-H₂), 1.83 (1H, m, 3′-H), 2.08 (2H, m, 17-H₂), 2.23 (2H,t-like, J=7.3, 2-H₂), 2.78 (2H, t-like, J=7.0, 14-H₂), 3.28 (2H, t-like,J=6.8, 11-H₂), 4.23 (1H, m, 2′-H), 4.55 (1H, m, 9-H), 5.29 (1H, m,15-H), 5.44 (1H, m, 16-H), 5.56 (1H, m, 13-H), 5.61 (1H, m, 12-H)

¹³C-NMR (100 MHz, CD₃OD): 12.4 (C-5′), 15.4 (C-18), 17.0 (C-6′), 22.3(C-17), 26.8, 27.1 (C-3,4), 27.5, 27.8 (C-4′,14), 31.0, 31.1, 31.2(C-5,6,7), 34.5 (C-8), 35.8 (C-2), 37.6 (C-3′), 39.2 (C-11), 77.6(C-2′), 78.6 (C-9), 123.0 (C-12), 127.7 (C-15), 129.2 (C-13), 134.6(C-16), 174.0 (C-1′), 176.6 (C-1), 203.9 (C-10)

Example 6 Study of Flower Bud Formation Promoting Activity of KetolFatty Acid Derivative II of the Invention in Morning Glory (Pharbitisnil)

Seeds (9 g) of morning glory (variety name: Murasaki) were treated withconcentrated sulfuric acid for 20 minutes, and then left to standovernight under running water. Subsequently, the treated seeds wereplaced on wet sea sand for 24 hours, hilum side up, to thereby formroots. The seeds having roots were planted in sea sand to a depth of 1.5cm to 2.0 cm, followed by culturing under continuous light (forapproximately 5 days).

The whole plants of the morning glory which had put forth leaves throughthe culture were transferred to a culture solution [KNO₃ (250 mg),NH₄NO₃ (250 mg), KH₂PO₄ (250 mg), MgSO₄.7H₂O (250 mg), MnSO₄.4H₂O (1mg), Fe-citrate n-hydrate (6 mg), H₃BO₃ (2 mg), CuSO₄.5H₂O (0.1 mg),ZnSO₄.7H₂O (0.2 mg), Na₂MoO₄.2H₂O (0.2 mg), Ca(H₂PO₄)₂.2H₂O (250mg)/distilled water (1,000 mL)].

While ketol fatty acid derivative (9) of the invention, which wasobtained in the above Example 5, or test compounds such as Compound (4)was directly sprayed onto the cotyledons of the above morning gloryplants grown in the culture system, the morning glory plants weresubjected to treatment in the dark, then raised under the application ofcontinuous light at 26° C. for 16 days. On day 16, the number of flowerbuds of the morning glory plants was counted (8 plants/group).

The treatment in the dark was performed overnight (for 14 hours).

In FIG. 6, along the X axis, (a) represents a control (a group of plantssprayed with distilled water), (b) represents a group of plants sprayedwith ketol fatty acid derivative (9) (1 μM), (c) represents a group ofplants sprayed with compound (4) (1 μM) and L-isoleusine (1 μM), (d)represents a group of plants sprayed with L-isoleusine (1 μM), (e)represents a group of plants sprayed with compound (4) (1 μM), and (f)represents a group of plants sprayed with compound (4) (100 μM). The Yaxis represents flower bud formation promoting activity (shown as thepercentage of each group to the control group (=100%) in terms of thenumber of flower buds per plant).

As shown in FIG. 6, ketol fatty acid derivative (9) of the inventionexhibits equal or higher flower bud formation promoting effect ascompared with compound (4), even when the ketol fatty acid derivative(9) has a lower concentration than compound (4). Since this effect wasnot observed in group (d) onto which only L-isoleucine was sprayed, theeffect is considered to be unique to amide derivatives such as ketolfatty acid derivative (9) of the invention.

FIG. 7 shows data used to determine the optimum administrationconcentration of ketol fatty acid derivative (9) of the invention. Theketol fatty acid derivative (9) was sprayed onto morning glory plants inthe same manner as that outlined above, at concentrations of 0 μM(control), 0.1 μM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM shown in theabscissa of FIG. 7 (8 to 24 plants were employed for one concentration),whereby the flower bud formation promoting activity (%) was studied.

As is shown in FIG. 7, the optimum administration concentration of ketolfatty acid derivative (9) of the invention was found to be approximately1 μM.

INDUSTRIAL APPLICABILITY

The present invention provides substances endowed with excellent plantgrowth regulating effect and processes for preparing the substances.Moreover, the invention provides plant growth regulators containing anyof the substances as the active component.

1. An α-ketol unsaturated fatty acid derivative represented by formula(1):

wherein R¹ represents linear C₁₋₅ alkyl; R² represents hydrogen,hydroxyl, methyl, or methyl, or ethyl; R³ represents hydrogen orcarboxyl; R⁴ represents hydrogen, methyl, ethyl, isopropyl, acetyl, orbtert-butyl; R⁵ represents hydrogen, hydroxyl, methoxy, ethoxy, formyl,or hydroxymethyl; n is an integer from 3 to 15; and the carbon atoms atthe A- and B-positions may be in either (R)- or (S)-configuration. 2.The α-ketol unsaturated fatty acid derivative as recited in claim 1,wherein R¹ in formula (1) is methyl.
 3. The α-ketol unsaturated fattyacid derivative as recited in claim 1, wherein n is
 7. 4. The α-ketolunsaturated fatty acid derivative as recited in claim 1, wherein thecarbon atoms at the A- and B-positions are in (S)- and(R)-configurations, respectively.
 5. The α-ketol unsaturated fatty acidderivative as recited in claim 1, wherein R⁵ in formula (1) is hydroxyl.6. The α-ketol unsaturated fatty acid derivative as recited in claim 1,wherein R² in formula (1) is hydroxyl, and R³ and R⁴ in formula (1) ishydrogen.
 7. The α-ketol unsaturated fatty acid derivative as recited inclaim 1, which is(9R)-11-{(2′S,8′S,10′S,11′R)-2′,8′-dihydroxy-7′-oxo-11′-[(Z)-2-pentenyl]-9′-oxa-4′-azatricyclo[6.3.1.0^(1.5)]dodec-5′-en-10′-yl}-9-hydroxy-10-oxoundecanoicacid.
 8. A process of preparing an α-ketol fatty acid derivative asrecited claim 1, comprising mixing an α-ketol unsaturated fatty acidrepresented by formula (2) with a catecholamine represented by formula(3) or a derivative thereof, and incubating the resultant mixture underneutral to basic conditions, to thereby yield an α-ketol unsaturatedfatty acid catecholamine derivative:

wherein R¹ and n have the same meanings as defined in claim 1, and

wherein R², R³, R⁴, and R⁵ have the same meanings as defined in claim 1,and R⁶ represents hydrogen, linear or branched alkyl, or acyl.
 9. Aprocess of preparing an α-ketol fatty acid derivative as recited inclaim 2, comprising mixing an α-ketol unsaturated fatty acid representedby formula (2) with a catecholamine represented by formula (3) or aderivative thereof, and incubating the resultant mixture under neutralto basic conditions, to thereby yield an α-ketol unsaturated fatty acidcatecholamine derivative:

wherein R¹ and n have the same meanings as defined in claim 1, and

wherein R², R³, R⁴, and R⁵ have the same meanings as defined in claim 1,and R⁶ represents hydrogen, linear or branched alkyl, or acyl.
 10. Aprocess of preparing an α-ketol fatty acid derivative as recited inclaim 3, comprising mixing an α-ketol unsaturated fatty acid representedby formula (2) with a catecholamine represented by formula (3) or aderivative thereof, and incubating the resultant mixture under neutralto basic conditions, to thereby yield an α-ketol unsaturated fatty acidcatecholamine derivative:

wherein R¹ and n have the same meanings as defined in claim 1, and

wherein R², R³, R⁴, and R⁵ have the same meanings as defined in claim 1,and R⁶ represents hydrogen, linear or branched alkyl, or acyl.
 11. Aprocess of preparing an α-ketol fatty acid derivative as recited inclaim 4, comprising mixing an α-ketol unsaturated fatty acid representedby formula (2) with a catecholamine represented by formula (3) or aderivative thereof, and incubating the resultant mixture under neutralto basic conditions, to thereby yield an α-ketol unsaturated fatty acidcatecholamine derivative:

wherein R¹ and n have the same meanings as defined in claim 1, and

wherein R², R³, R⁴, and R⁵ have the same meanings as defined in claim 1,and R⁶ represents hydrogen, linear or branched alkyl, or acyl.
 12. Aprocess of preparing an α-ketol fatty acid derivative as recited inclaim 5, comprising mixing an α-ketol unsaturated fatty acid representedby formula (2) with a catecholamine represented by formula (3) or aderivative thereof, and incubating the resultant mixture under neutralto basic conditions, to thereby yield an α-ketol unsaturated fatty acidcatecholamine derivative:

wherein R¹ and n have the same meanings as defined in claim 1, and

wherein R², R³, R⁴, and R⁵ have the same meanings as defined in claim 1,and R⁶ represents hydrogen, linear or branched alkyl, or acyl.
 13. Aprocess of preparing an α-ketol fatty acid derivative as recited inclaim 6, comprising mixing an α-ketol unsaturated fatty acid representedby formula (2) with a catecholamine represented by formula (3) or aderivative thereof, and incubating the resultant mixture under neutralto basic conditions, to thereby yield an α-ketol unsaturated fatty acidcatecholamine derivative:

wherein R¹ and n have the same meanings as defined in claim 1, and

wherein R², R³, R⁴, and R⁵ have the same meanings as defined in claim 1,and R⁶ represents hydrogen, linear or branched alkyl, or acyl.
 14. Aprocess of preparing an α-ketol fatty acid derivative as recited inclaim 7, comprising mixing an α-ketol unsaturated fatty acid representedby formula (2) with a catecholamine represented by formula (3) or aderivative thereof, and incubating the resultant mixture under neutralto basic conditions, to thereby yield an α-ketol unsaturated fatty acidcatecholamine derivative:

wherein R¹ and n have the same meanings as defined in claim 1, and

wherein R², R³, R⁴, and R⁵ have the same meanings as defined in claim 1,and R⁶ represents hydrogen, linear or branched alkyl, or acyl.